Depletion of ovarian follicles is associated with the end of reproductive function in ageing females. Recently, it has been described that this process parallels increases in the concentration of norepinephrine (NE) in the rat ovary. In sexually mature rats, experimentally-induced increases in the sympathetic tone of the ovary is causally related to ovarian cyst formation and deranged follicular development. Thus, there is a possibility that increased ovarian NE concentrations represent changes in the activity of sympathetic nerves, which consequently participate in the process of ovarian cyst formation observed during ageing in the human and experimental animal models.3H-NE recently incorporated under transmural depolarisation in relation to changes in the ovarian follicular population. Morphometric analysis of ovarian follicles and real time PCR for Bcl2 and Bax mRNA were used to assess follicular atresia.Sprague-Dawley rats between 6 and 14 months old were used to analyse the capacity of the ovary to release 3H-norepinephrine (3H-NE) from the ovary and ovarian NE concentrations increased, reaching their peak values at 12 months old and remained elevated up to 14 months old. Increases in sympathetic nerve activity paralleled changes in the follicular population, as well as disappearance of the corpus luteum. In contrast, luteinised follicles, precystic follicles, and cystic follicles increased. During this period, the relationship between Bax and Bcl2 mRNAs  increased, suggesting atresia as the principal mechanism contributing to the decreased follicular population. When NE tone was increased, the mRNA ratio favoured Bcl2 to Bax and antiapoptotic signals dominated this period of development. Thus, these changing ratios could be responsible for the increase in luteinised follicles, as well as precystic and cystic follicles.From 8 months old, the induced release of recently incorporated These data suggest that the ageing process in the ovary of the Sprague-Dawley rat is accompanied by an increased sympathetic tone of the ovary. Consequently, this sympathetic change could be related to a neuroendocrine-driven formation of a polycystic condition similar to that observed in the sympathetic-activated adult ovary. Reproductive senescence in mammals is a poorly understood process. Moreover, understanding the mechanism underlying the loss of ovarian function is especially important because of an increasingly aged population and the fact that pregnancy has been postponed continuously during recent decades ,2. DiverThere is little information regarding the role of sympathetic activation within the ovary of middle aged animals and the potential involvement in the loss of reproductive function. If the recently reported increase in NE concentration  is relat3H-NE release and for morphology experiments (one ovary for each technique either from the left or right side). The other 5 rats per group were used to measure endogenous ovary NE content, as well as for RNA extraction and real-time PCR (one ovary for each technique either from the left or right side). Table Adult female Sprague-Dawley rats derived from a stock kept at the University of Chile were used. Rats from 6 to 14 months old were maintained in individual cages at 20–23°C in an alternating 12 h light:dark regimen (lights off from 1900 h to 0700 h) with food and water ad libitum. Forty-five rats were used, which were divided into the following five age groups with 9 rats per group: 6, 8, 10, 12 and 14 months old. Four rats per group were used to examine All animal procedures were performed following protocols previously approved by the Institutional Ethic Committee of the Faculty of Chemical and Pharmaceutical Sciences, Universidad de Chile, and experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction. The animals were sacrificed by decapitation, trunk blood was collected, and serum was stored at 4°C in order to determinate progesterone, androstenedione, and estradiol levels. The ovaries were collected and immediately frozen at -80°C, with the exception of the four left-sided ovaries in each group that were fixed for morphological studies. In each rat, the stage of the estrual cycle was determined by microscopic analysis of the predominant cell type obtained from daily (Monday through Friday) vaginal smears. All rats were sacrificed during diestrus.Fresh ovaries were immersed in Zamboni fixative, embedded in paraffin, cut into 6 um sections, and stained with hematoxylin and eosin. The presence of preantral, antral, atretic, precystic (type 3), and cystic follicles was analysed according to Lara et al. . All sliSerum estradiol, androstenedione, and progesterone levels were measured by an enzyme immunoassay (EIA) following the manufacturer's instructions . Estradiol sensitivity was 10 ng/ml, and the intra- and inter-assay variations were less than 9.3% and 9.9%, respectively. Progesterone sensitivity was 0.1 ng/ml, and intra- and inter-assay variations were less than 10.6% and 12.6%, respectively. Androstenedone sensitivity was 0.05 ng/ml, and the intra- and inter-assay variations were less than 6.7% and 14.6%, respectively.The adrenal glands were weighed and homogenised in 500 μL of ice cold 0.2 N PCA. The homogenate was centrifuged at 12,000 g for 10 min at 4°C. The supernatants were used for colourimetric determination of total catecholamines. As previously reported , this me4 and centrifuged . NE was measured in the supernatant by HPLC coupled with electrochemical detection [2PO4, 0.42 mM octyl-sulphate, 0.02% EDTA and 1.5% acetonitrile (pH 2.5) with a 0.9 ml/min flow rate. The potential of the amperometric detector was set to 0.7 V. Under these experimental conditions, the retention time was 4 min for NE and 10 min for DHBA.One frozen ovary from each rat was homogenised in 200 μl 0.1 M acetic acid. Subsequently, 50 μl of the homogenate was precipitated with 180 μl 0.2 M HClOetection . A 150 μ2-5%CO2, and then incubated for 30 min at 37°C with 2 μCi 3H-NE . After washing the tissue in KRB (6 washes of 10 min each) to remove non-incorporated radioactivity, the ovaries were transferred to a thermoregulated superfusion chamber and perifused at a 1.5 ml/min flow rate for 10 min with KRB plus 10 mM tetraethylammonium, a potassium channel blocker which enhances the release of NE from nerve terminals in response to electrical stimulation [3H-catecholamine remaining in the tissue was determined by scintillation counting in a Tri-Carb Liquid Scintillation Analyzer 1600TR , with 72.5% efficiency for 3H. These values were used to calculate the radioactivity remaining in the tissue after the experiment. In order to calculate the amount of NE released, the radioactivity present in each of the one-minute samples was determined in the same way. The release, which represented 3H-NE overflow from the ovaries, was then expressed as fractional release, i.e. as a percentage of the total radioactivity present in the tissue. The total amount of neurotransmitter released was calculated as the area under the curve after stimulation minus that for basal efflux [The procedure was performed as previously described ,23, withmulation ,25. One-l efflux ,24.Total RNA was extracted as described by Chomczynski and Sacchi  from who2, 0.16 μM each primer and 2 μl cDNA. After initial polymerase activation at 95°C for 10 min, primer-specific amplification and quantification cycles were run at 60°C for 15 s and 72°C for 20 s. The fluorescence intensity of the double-strand specific SYBR-Green I, reflecting the amount of PCR-product actually formed was read at the end of each elongation step, after previous melting curve analyses to determine the melting points of the PCR products. Then, the amounts of specific initial template mRNA were calculated by determining the time point at which the linear increase of sample PCR product started relative to the corresponding points of a standard curve, which was obtained by serial dilutions of known copy numbers of the corresponding control tissue. To evaluate specific amplification, a final melting curve was created (72–95°C) under continuous fluorescent measurement.For specific gene amplification, a standard protocol of 40 cycles was used in a MJ Research PT-200 . A standard real-time PCR reaction mix was prepared containing the following components: 15 μl of Platinum SYBR Green PCR Super MIX UDG , 2.4 mM MgClBcl2 mRNA (Genebank access NM_016993). The Bax primers were composed of a forward primer (5-AGG-CGA-ATT-GGC-GAT-GAA-CTG-3) and reverse primer (5-TTC-TGA-TCA-GCT-CGG-GCA-CTT-TAG-3) (Genebank access U49729). These primers were from Invitrogen/Life Technologies, Inc. To normalise the quantification of Bcl2 and Bax mRNAs, ribosomal 18s mRNA was measured in each protocol. Amounts of 18s mRNA were determined using a commercially available RT primer pair . Amplification of 18s mRNA was performed in a different tube to avoid interference with the amplification of the mRNAs. Reaction tubes lacking RT were used as PCR controls. The RT-PCR products were separated on 2.0% agarose gels, stained with ethidium bromide, and photographed digitally.The primers used for amplification were a forward primer (5-CAC-CCT-GGC-ATC-TTC-TCC-TT-3) and reverse primer (5-ACA-CAT-GAC-CCC-ACC-GAA-CTC-3) corresponding to Differences between the different ages groups were analysed by one-way ANOVA, followed by the Student-Newman-Keuls multiple comparison test for unequal replication. The level of significance was set to P < 0.05.The morphological analysis of the ovary Figure  showed iAs shown in Figure As shown in Figure As shown in Table 3H-NE was measured. The results demonstrated a constant increase in the stimulation-induced release capacity of the ovary from 8 months old, reaching a peak at 12 months  developed a similar morphological change in the ovary similar to what we have described in the ovaries of ageing rats. These morphological changes were chronologically related with a previous increase in nerve activity (as measured by NE concentration), and with the capacity of the ovary to release recently incorporated stimulus ,13. All stimulus , periphestimulus ,13,40, wstimulus  demonstrSupporting this assertion, it has been demonstrated  that ovaAltogether, these data suggest that an increased intraovarian sympathetic tone participates in the processes that induce the polycystic condition associated with ageing in the Sprague-Dawley rat, and it has opened a new mechanism to control follicular development during natural ageing. The use of rats as a model to investigate the natural ageing of ovary function appears to be a useful model for studying the processes and mechanisms which control follicular development.The authors declare that they have no competing interests.EA: quantification of catecholamines in adrenal gland and NE in celiac ganglion and analysed of reproductive parameters of the rats RF: chromatographic determination of adrenalin in plasma and ovary. DF: primer design and determination of Bcl2 mRNA by RT-Real Time PCR. MPG: primer design and determination of Bax mRNA by RT-Real Time PCR. MG: noradrenalin release determination. HL: discussion and manuscript preparation. AP: participated in ovarian morphology analysis, noradrenalin release determination, and contributed to the development, design, coordination of the research, manuscript preparation and was responsible for the grant. All authors read and approved the final manuscript.


Genes of this family encode proteins containing a highly conserved bHLH domain, which take part in the regulation of the development of central nervous system and peripheral nervous system. Many AS-C homologs have been isolated from various vertebrates and invertebrates. Also, AS-C genes are duplicated during the evolution of Diptera. Functions besides neural development controlling have also been found in Drosophila AS-C genes.achaete-scute homologs (ASH) from the lepidopteran model organism Bombyx mori, including three proneural genes and one neural precursor gene. Proteins encoded by them contained the characteristic bHLH domain and the three proneural ones were also found to have the C-terminal conserved motif. These genes regulated promoter activity through the Class A E-boxes in vitro. Though both Bm-ASH and Drosophila AS-C have four members, they are not in one by one corresponding relationships. Results of RT-PCR and real-time PCR showed that Bm-ASH genes were expressed in different larval tissues, and had well-regulated expressional profiles during the development of embryo and wing/wing disc.We cloned four achaete-scute homologs in Bombyx mori, the second insect having four AS-C genes so far, and these genes have multiple functions in silkworm life cycle. AS-C gene duplication in insects occurs after or parallel to, but not before the taxonomic order formation during evolution.There are four  Firefly luciferase activity and Renilla luciferase activity were determined with 20/20n Luminometer . Each treatment was repeated at least three times.Promoter segments were cloned into Luciferase Reporter Vector pGL3-Basic separately, and the ORF region of silkworm enhancer . Then thenhancer . 0.1 μg pfu DNA polymerase.Primers P-F and P-R were designed at the terminals of the target segment sequence. Reversed primers m-R and m-F with the mutated bases were designed around the site where the point mutation would be introduced. PCRs were carried out with P-F pairing m-R or m-F paring P-R. After purification, the two PCR products were mixed, and denatured, renatured and extended for three cycles without any primers. Three cycles after running, P-F and P-R were added and the PCR was continued for 30 cycles of amplification. All of the PCRs were processed with ASH genes expression during embryo development and wing development. Primers were designed based on the cDNA sequence and a segment around 200 bp would be specially amplified for each gene. The housekeeping gene Bm-actin A3 was used as the internal control. A 20 μl volume containing cDNA produced from about 10 ng of total RNA, 5 pmol of each primer, and 10 μl of SYBR Green Realtime PCR Master Mix  was used for each PCR reaction. Then the PCR was processed on a Chromo4 Four-Color Real-Time System (Bio-Rad [formerly MJ Research]) under the following program: one cycle of 95°C for 3 min; then 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 30 sec. The melting curve was established from 60°C to 95°C. Three independent repeats were carried out for each reaction. Threshold cycle values were used for the further analysis.Q-PCR was used to determine the changes of silkworm 6 to 102 copies. The data curve and the melting curve showed the reliability and specificity of the PCR product . The log ranges of the standard curves were from 10uct Fig. . ThreshoBombyx ASH genes during the development course of silkworm embryo and pupal wing were constructed.Theoretic copy of each sample was calculated with the linear equation for each gene, and trendlines for Bm-ASH1 and helped write the paper. YLL took part in RT-PCR and helped analyzing the data. YYZ cultured the cells. XWH and ZZF conceived the project and draft the manuscript. All authors read and approved the final manuscript.ZQX carried out most of the work and wrote the paper. ZTY isolated


For more than three decades, diseases caused by salmonid alphaviruses (SAV) have become a major problem of increasing economic importance in the European fish-farming industry. However, experimental infection trials with SAV result in low or no mortality i.e very different from most field outbreaks of pancreas disease (PD). This probably reflects the difficulties in reproducing complex biotic and abiotic field conditions in the laboratory. In this study we looked at the relationship between SAV-infection in salmon and sub-lethal environmental hypoxia as a result of reduced flow-through in tank systems.The experiment demonstrated that constant reduced oxygen levels (60-65% oxygen saturation: 6.5-7.0 mg/L) did not significantly increase the severity or the progress of pancreas disease (PD). These conclusions are based upon assessments of a semi-quantitative histopathological lesion score system, morbidities/mortalities, and levels of SAV RNA in tissues and water . Furthermore, we demonstrate that the fish population shed detectable levels of the virus into the surrounding water during viraemia; 4-13 days after i.p. infection, and prior to appearance of severe lesions in heart (21-35 dpi). After this period, viral RNA from SAV could not be detected in water samples although still present in tissues (gills and hearts) at lasting low levels. Lesions could be seen in exocrine pancreas at 7-21 days post infection, but no muscle lesions were seen.In our study, experimentally induced hypoxia failed to explain the discrepancy between the severities reported from field outbreaks of SAV-disease and experimental infections. Reduction of oxygen levels to constant suboptimal levels had no effect on the severity of lesions caused by SAV-infection or the progress of the disease. Furthermore, we present a modified VIRADEL method which can be used to detect virus in water and to supplement experimental infection trials with information related to viral shedding. By using this method, we were able to demonstrate for the first time that shedding of SAV from the fish population into the surrounding water coincides with viraemia. Alphavirus, Togaviridae) have become an increasing problem of economical importance to the European fish-farming industry. Salmonid alphavirus (SAV) is the only alphavirus that has been isolated from fish, and are thought to comprise at least six subtypes (SAV1-6) )20 water. The Verso™ 1-step QRT-PCR Rox kit from Thermo Scientific was used for real-time RT-PCR analysis. The nsP1-assay targeting the nsP1-gene in SAV [A) assay [H. salinarum  was used as exogenous controls when extracting RNA from plasma and water samples. The Sal-assay generates a 59 bp PCR-product (position 541-600 of Acc.# AB219965). The real-time master mixture consisted of 6.25 μl 1-step QPCR Rox Mix (2 ×), 0.625 µl RT Enhancer, together with 0.125 µl of Verso Enzyme mix. Primer and probe concentrations had been optimized for each assay; F primer/R primer/probe: nsP1 assay (SAV): 900 nm/900 nm/200 nm, EF1AA assay ; 900 nm/900 nm/225 nm), Sal assay: 300 nm/900 nm/200 nm, VHSV08 assay: 600 nm/600 nm/225 nm. Primers and probes at their respective concentrations were added to the master mixture and adjusted with ddH20 to a total volume of 10.5 µl prior to adding 2 µl of RNA template. The real-time RT-PCR reaction was run in a 7500 Fast Real-Time PCR System cycler from Applied Biosystems using the following conditions: reverse transcription at 50°C for 15 minutes followed by activation of the Thermo-Start DNA polymerase at 95°C for 15 minutes prior to amplification with 45 cycles of 95°C for 15 seconds and 60°C for 1 min . For each assay a standard curve was generated from dilution series of RNA in 20 ng/μl yeast tRNA (Invitrogen)  or ddH20 (VHSV-assay).Total RNA was extracted from tissues (gills and hearts 10-20 μg) using TRIreagent (Sigma) according to the method described by Devold and coworkers , wherease in SAV  was applA) assay  were useA) assay  and the A-assay (tissues) whereas plasma samples were normalized against the VHSV08-assay. Water samples were normalized against both the Sal-assay and the VHSV08-assay. Samples from dead fish were not normalized, but only considered as SAV-positive or SAV-negative. RNA extraction controls and no template controls (NTC) were included in all runs in order to detect possible contamination. In addition, a positive control was included in all runs in order to detect reagent mix errors. If water or plasma samples were found SAV-positive, one parallel sample that had not been spiked with VHSV was also checked for the presence of VHSV that could have given rise to false background for normalization of real-time RT-PCR data (VHSV-negative controls).All samples that were analyzed with real-time RT-PCR were performed in triplicate. Only samples that were positive in triplicates were considered for normalization. Thresholds for all assays were set to 0.01 except for the Sal-assay which was set to 0.001. Ct-values obtained for the target gene (nsP1-assay) were normalized against the endogenous control EF1AA)- and exogenous reference genes  by the use of the Microsoft®Excel® based computer software Q-Gene [Mean Ct-values for the target gene nsP1 were normalized against endogenous . Pictures were taken with a digital Olympus camera E-330 or a Nikon DS-US1 camera with NIS-Elements software version 5.03 (Nikon Instruments Inc). Histopathological lesions in pancreas, muscle and hearts were evaluated from five fish from each group at each time point from the SNorm and SRed groups (n = 60), whereas three fish from each group at each time point were evaluated from CNorm and CRed (n = 40). Lesions were scored in accordance to a semi-quantitative lesion score system  from five fish from all experimental groups at six time points were fixed in a modified Karnovsky's fixative containing Ringer's solution with 4% sucrose, and kept at 4°C until further processing. Tissues were washed 3 times (15 min each) in a phosphate buffer with Ringer's solution and dehydrated in an ethanol series (70%-96% ethanol). The tissue were then infiltrated with Historesin (7022 31731 Leica Historesin Embedding Kit) (Leica Microsystems) or with Technovit 7100 (Hereaus Külzner) as described by the manufacturer, and left to harden in molds over night at room temperature. Sections 1.5-2 μm thick were cut on a Reichert- Jung 2050 Supercut microtome (Cambridge Instruments) or a Leica RM2255 and then mounted on slides in dHThe PCR efficiency, regression analysis and standard curve slope s  of the various assays were calculated from the Ct-values obtained from dilution series of RNA and are given in Table Idiomarina sp., Cobetia marina, Janibacter sp., Bacillus sp., Tenacibaculum/Flavobacterium, Vibrio sp., Vibrio splendidus, and Pseudoalteromonas sp.Several bacteria were isolated on marine agar and blood agar (2% NaCl) from head kidney of salmon during this experiment. By BLAST search tool the bacteria were identified to genus and it was established that they all belonged to marine genera; 2 per liter, approx. 300-400 lh-1tank-1) to 60-65% saturation  by reduction of the flow-through rate. The average oxygen level during the experiment in the various experimental tanks was; CNorm; mean 86.29 , CRed; mean 64.79, , SNorm; mean 84.32 , SRed; mean 65.57 , respectively.Two days after infection, the oxygen levels in one tank with uninfected fish (CRed) and one tank with SAV-infected fish (SRed), were gradually (slowly within a 24 h period) lowered from 85-90% saturation  to 12.3% (CRed) in the uninfected controls, and from 1.5% (SNorm) to 10.8% (SRed) in the SAV-infected groups , especially of the dorsal and pectoral fins, could be seen in all experimental groups throughout the study. Some individuals also had petecchial bleedings/erythemia on abdomen and at pectoral fin bases, and four dead fish had severe erosions behind pectoral fins (CRed and SRed groups).During the 70 days the experiment lasted, it was not possible to see a significant increase in mean body length or weight for the fish groups, nor a considerable difference in mean weight, length or Fulton's condition factor (K) between the various groups  Figure . This noA were not significantly different between the SAV-infected groups in gills or hearts during the experiment.No SAV viral RNA could be detected in gills from the uninfected groups reared at normal or reduced oxygen levels. However, in hearts from the same individuals, very low amounts of SAV viral RNA could be detected in 7 out of 60 fish at 7-21 dpi. The raw Ct-values were in the range of 32-36. In both SAV-infected groups, viral RNA from SAV was detected in gills Figure  at all sH. salinarum and the RNA-extraction control VHSV, it was evident that the highest amounts of viral RNA in water in both SAV-infected groups could be seen at 6 days post infection, declining at 10-13 dpi  of the examined individuals from the control groups. No muscle lesions were found in any of the experimental groups throughout the study. Two moribund fish were sampled from CNorm at 56 and 62 dpi, and one fish from SRed at 42 dpi. Pancreas, heart and muscle tissue were processed for histology and examined, with no lesions recorded in these organs.Vacuolation and rounding of the acinar cells could be seen in 5 out of 8 individuals at 7 dpi.  in field versus experimental infections with SAV. It is possible that certain key environmental factors, such as oxygen levels, might play an important role concerning the severity or the progress of PD. In general, hypoxia can act as a stressor to fish  which maThe SAV-infection led to severe lesions in hearts during the course of infection, characterized by epicarditis and multifocal degeneration of cardiomyocytes in ventricle of the heart. Ferguson et al  concludeSalmon has been shown to produce protective neutralizing antibodies shortly after i.p infection , readilySalmo salar L. smolts that were intraperitoneally (i.p.) infected with a salmonid alphavirus (SAV3) isolate (SAVH30/04) shed detectable levels of virus into the surrounding water in the period 4-13 days post infection. This was assessed by a VIRADEL (virus-adsorption-elution) method using electropositive 1MDS virus filters . The. TheSalmers , length in cm and weight in grams for all groups during the experiment.Click here for fileLength. The additional files K-factor.jpg, length.jpg and weight.jpg describe mean development of condition factor (K), length in cm and weight in grams for all groups during the experiment.Click here for fileWeight. The additional files K-factor.jpg, length.jpg and weight.jpg describe mean development of condition factor (K), length in cm and weight in grams for all groups during the experiment.Click here for file


Systemic lupus erythematosus (SLE) develops in relation to many environmental factors. In our opinion, it is more important to investigate the effect of melatonin on the environmental- related SLE. In the present study, 0.5 ml pristane were used to induce SLE in female BALB/c mice. Melatonin  was orally administered immediately after pristane-injection for 24 weeks. IgM anti ssDNA and histone antibodies were detected after 0, 1, 2, 4, 8 weeks pristane injection. The levels of IL-2, IL-6 and IL-13 were detected after 24 weeks. Renal lesions were also observed. The results showed that melatonin antagonized the increasing levels of IgM anti ssDNA and histone autoantibodies. Melatonin could also decrease the IL-6 and IL-13 production and increase the IL-2 production. Besides, melatonin could lessen the renal lesions caused by pristane. These results suggested that melatonin has a beneficial effect on pristane-induced lupus through regulating the cytokines disturbances. These cytokines have close relations to the development of SLE such as autoantibodies production and immune-complex nephritis. But there are some contradictory results about the changes of some cytokines in different reports. Systemic lupus erythematosus (SLE or Lupus) is an autoimmune disease that can attack the body's normal tissue and cells, resulting in inflammation and tissue damage , 2. SLE In recent years, more and more attention has been paid to environmental factors that may be implicated in the pathogenesis of SLE , 7. AutoMelatonin is synthesized and secreted mainly by the pineal gland that can make specific receptors in and out of the central nervous system. Melatonin not only can directly affect inflammation and immune cells, but also has indirect influences on it through thalamencephalon . More imTo investigate the role of melatonin in SLE, especially in the environmental-related lupus, pristane-induced mice were used. The effects of melatonin on the cytokine disturbances and the following changes in pristane-induced SLE model were also determined. Sixty female BALB/c mice aged two months (17 ± 2 g) were supplied by the Experimental Animal Center of Anhui Medical University. All experimental protocols described in this study were approved by the Ethics Review Committee for Animal Experimentation of Institute of Clinical Pharmacology, Anhui Medical University.ABC. Mouse interleukin-2 ELISA kits and interleukin-6 ELISA kits were purchased from ADL. Mouse interleukin-13 ELISA kits were purchased from BIOO. Melatonin was purchased from Sigma. Pristane, heat-denatured calf thymus DNA (ssDNA), total calf thymus histone (histone), concanavalin A (ConA), and lipopolysaccharides (LPS) were also from Sigma. Biotin-conjugated rabbit-anti-mouse IgM antibodies and horseradish peroxidase-labeled avidin were purchased from S Sixty female BALB/c mice were randomly divided into six groups: normal control group, model group, prednisone-treatment group which served as positive control group, and melatonin  treatment groups including melatonin group one (MT1), melatonin group two (MT2), and melatonin group three (MT3). The mice in normal control group were given an intraperitoneal injection of 0.5 ml normal saline (NS), and the other groups were given an intraperitoneal injection of 0.5 ml pristane. The mice in normal control group and model control group received intragastric administration of NS per day after the first treatment. The mice in positive control group were given intragastric administration of 5 mg/kg prednisone (Pre) per day. The mice in MT1 group were intragastrically treated with 0.01 mg/kg melatonin, MT2 0.1 mg/kg melatonin, and MT3 1.0 mg/kg melatonin. Sera were collected from the tail vein before treatment (0) and 2, 4, and 8 weeks after treatment to measure the level of autoantibodies. Twenty-four weeks after pristane administration, all mice were killed, and spleens were removed for immunological detections; meanwhile, nephric tissues were examined by light microscopy.μL of substrate —tetramethylbenzidine (TMB) and hydrogen peroxide solution were added to the wells for 30 minutes at 37°C. The reaction was terminated by addition of 50 μL of 2 M H2SO4 per well. The intensity of the yellow color was read at 490 nm in a microplate reader. The sera of other eight normal BALB/c mice were collected and detected at the same time, and the mean density and SD were calculated to serve as control. The mean enzyme indexes (EI) of the samples in different groups were calculated as: Anti-ssDNA and histone IgM antibodies were detected by an ELISA technique similar to the one as described in . 96-well6 cells/ml. The cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 10 ml penicillin, streptomycin and antimycotic solution/L (Sigma). Cultures were stimulated with 3 mg/L ConA, 12 mg/L LPS, or medium alone and incubated at 37°C in humidified air with 5% CO2. The supernatant was harvested at 48 h and preserved at −20°C for cytokine assays. Spleen cell Culture and induction of cytokines were modifications of the methods as described in , 19. Bri The concentrations of interleukin-2 (IL-2), interleukin-6 (IL-6), and interleukin-13 (IL-13) in the supernatant were determined using appropriate commercial ELISAs for the murine form of these cytokines. The intensity of each sample was read at 450 nm in a microplate spectrophotometer. The kidneys collected while sacrificing mice were fixed for 24 hours in neutral buffered formalin prior to paraffin embedding. Sections were stained with Haematoxylin and Eosin and Jones silver methenamine and then examined under a light microscope for the severity of the renal lesion. Glomerulonephritis, renal tubular lesions and interstitial inflammation were observed, and the degree of severity of histological lesions characteristic of lupus nephritis was evaluated as absent (−), mild (+), moderate (++), and severe (+++). t-test with P < .05 as the minimal level of significance Results were expressed as the mean ± SD. Statistical analysis was performed using Student's two-wayP < .05, Figures The levels of IgM anti-ssDNA and histone antibodies were significantly different between pristane-injection and melatonin treatment groups (P < .05). In MT2 group, the level of anti-ssDNA IgM antibodies in sera increased during the first four weeks, but much lower than that of model group (P < .05), and remained normal in other periods. In MT3 group, the level of anti-ssDNA IgM antibodies did not increase  and were back to normal at 4 wk. In MT2 and MT3 groups, the levels of antibodies increased during the first four weeks, but much lower than model group (P < .01), and remained normal in the other periods , while IL-6 and IL-13 production of splenocytes from mice in model control group was higher than that from normal mice (P < .01). In MT2 and MT3 groups, IL-2 levels were up to the normal level and higher than that of model mice obviously (P < .05), while the IL-6 and IL-13 levels were lower than that of the model mice obviously   .The renal histopathological changes of the mice are shown in As shown in As shown in Figures There were some improvements of different degrees in melatonin-treated mice . GlomeruIn this study, the immune disturbances of pristane-primed mice and the effects of melatonin were observed. Since anti-DNA and histone antibodies were sensitive in SLE , 21, we in vitro experiments, melatonin could inhibit the immune effects of some cytokines and T lymphocytes [Cytokines network plays an important role in the development of SLE. In some phocytes . So we ophocytes . So the IL-2 is secreted by Th1-cells, and IL-13 is secreted by Th2-cells. There are some contradictory results in the documents about the changes of IL-2 and IL-13. So in this pristane-induced SLE model, we investigated the levels of the two cytokines. Our results revealed that the level of IL-2 in the pristane-injected mice was lower than that in the normal mice, while the level of IL-13 in the pristane-injected mice was higher than that in the normal mice. We also found that melatonin could decrease the IL-13 production but increased the IL-2 production. Many reports showed that the level of IL-2 decreased in the SLE, especially in the active stage, and that IL-2 could down-regulate the B lymphocytes activation . Some re4+ T cells and significantly promote the Th1-type cytokines, which participate in regulating the immune disturbances [The decreasing level of Th1-type cytokine and the increasing levels of Th2-type cytokine also indicated that there was a shift from Th1 towards Th2 in pristine-induced lupus. Some reports showed exogenous melatonin could activate the CDurbances . Our resPrednisone, which is a common drug for SLE , was serIn conclusion, pristane-induced lupus displays disturbance in immune systems, such as high level of autoantibodies and imbalance of cytokine network, which induces the followed nephritis. The changes are similar to the human lupus, and pristane is common in the environment; thus this model is appropriate to study the effects of melatonin on environmental-related SLE. On the other hand, we also demonstrated that the administration of melatonin inhibited the changes in the pristane-induced lupus. And that suggested melatonin had a beneficial effect on environmental-related lupus. The mechanisms might involve its modulation of disturbances in the immune system, especially in cytokines network. However, further studies should be necessary for a better understanding of the regulating process and the key factor for its action on autoimmune diseases.


Aggregation inhibitors are expected to reduce α-synuclein cytotoxicity, and an aggregation accelerator has recently been reported to reduce α-synuclein cytotoxicity. Therefore, amyloid aggregation modulating ligands are expected to serve as therapeutic medicines.α-Synuclein is a Parkinson's-disease-related protein. It forms aggregates in silico panning, a method which we have proposed previously. In this study, we selected as the target a very hydrophobic region known as the amyloid-core-forming region. Since this region cannot be dissolved in water, it is difficult to carry out the in vitro screening of its peptide ligand. We carried out 6 rounds of in silico panning using a genetic algorithm and a docking simulation. After the in silico panning, we evaluated the top peptides screened in silico by in vitro assay. These peptides were capable of binding to α-synuclein.We screened peptide ligands against α-synuclein by in silico panning. The screened peptides bind to α-synuclein, thus affecting the aggregation of α-synuclein.We demonstrated that it is possible to screen α-synuclein-binding peptides by  Lewy bodies are inclusion bodies observed in Parkinson's-disease patients. The major component of the Lewy body is amyloid-like fibrils of α-synuclein . The famin vitro . Especiain vitro . Therefoin vitro -11. Howein vitro . Recentltoxicity . Thus, aTo obtain an effective peptidic aggregation inhibitor, the peptide should bind to the region that plays an important role in amyloid fibril formation. The hydrophobic central region of α-synuclein called the "non-amyloid-β component of the amyloid plaque" (NAC) (residues 61–95) is the most important region in the formation of amyloid-like fibrils. In particular, some of the partial peptides in NAC, such as α-synuclein 68–78 (GAVVTGVTAVA), form amyloid-like fibrils by themselves . We haven), it is not sufficient to screen the huge sequence space randomly to obtain target peptides. In a previous study, we have reported an evolutional screening method using genetic algorithms (GAs) [in silico panning [in silico panning is shown in Figure Since N-mer peptides have a huge sequence variety (20ms (GAs) -18. GAs ms (GAs)  and the ms (GAs) ; these s panning . The schin silico panning in order to screen the peptides which bind to the α-synuclein's amyloidogenic region and affect α-synuclein fibril formation.In related studies, ADAPT and ENPDA have been reported ,24. ADAPWe selected an 11-mer peptide, α-synuclein 68–78 (GAVVTGVTAVA), as the target region. Although the three-dimensional structure of α-synuclein has been reported (1XQ8 ), this s4 = 10,000. However, even a docking simulation of all 10,000 sequences would be difficult because a peptide docking simulation requires more time than the docking simulation of a small compound. We therefore applied a genetic algorithm to the peptide in silico screening.Since we have succeeded in screening peptide ligands by using a tetramer peptide in the previous study, we selected a tetramer peptide in this study as well. To carry out effective screening using a genetic algorithm, we restricted the type of amino acids composing the peptides. In this study, since the target region was very hydrophobic, we selected the hydrophobic amino acids and the polar amino acids that tended to form hydrogen bonds , and the total diversity is 10st to the 3rd round, the docking energy decreased through in silico panning . The best peptide was obtained in the 6th generation. Table th generation. The peptides were ranked by their intermolecular energy. The top peptides had many glutamine residues that can form hydrogen bonds. Some peptides had a few hydrophobic residues, as calculated by Generalized Born/Surface Area in a continuous solvent model.First, we evaluated the peptides by their docking energy: the sum of the intermolecular energy, the intramolecular energy of the ligand and the desolvation energy. Although the intramolecular energy is not related to the binding affinity, we included this energy to eliminate unstable ligands. From the 1g Figure . As expeThe affinities of the screened peptides were determined by surface plasmon resonance measurement. The screened peptides were immobilized on a CM5 sensor chip by amine coupling. In this study, we evaluated QSTQ, GSQQ, SQTQ and AQTQ which were the top peptides, and PTYF, which was an inferior peptide. The N-terminal glutamine could have formed pyroglutamine, and the N-terminal proline did not have a primary amine. Thus, we added an alanine residue to the N-terminals of these peptides to immobilize them on the CM5 sensor chip and evaluated them. The dissociation constants were determined using a Scatchard plot. We were able to confirm the peptide binding to α-synuclein, and the KD value of QSTQ was determined to be 19 μM. AQTQ also bound to α-synuclein with similar affinity of QSTQ. GSQQ and SQTQ have lower affinity than QSTQ and AQTQ. In the case of PTYF, its KD was more than 10 times that of QSTQ. Since docking simulation still has problems inaccuracy, we have to evaluate several peptides to find good ligands. In this study, we can obtain the high affinity peptide by assaying the top 4 peptides.To determine the effect of peptides on α-synuclein aggregation, we co-incubated α-synuclein with 5-fold molar excess of the screened peptides. The α-synuclein fibril amounts were evaluated by Thioflavin T (TfT) binding assay. It is known that when TfT binds to amyloid fibrils, the fluorescence intensity around 480 nm increases. The α-synuclein solution without peptide showed an increase in the fluorescence intensity of TfT after 40 h of shaking Figure . On the et al. have proposed a stochastic approach to reduce the number of evaluated peptides [in silico panning," and have previously reported the screening of a glucose dehydrogenase noncompetitive inhibitor. For the recognition of the target protein, peptides need to have functional amino acids, and the amino-acid sequence is more important than the position of each amino acid. In some cases, the combination of independent important amino acids results in loss of binding affinity. The GA can search the sequence space by optimizing the amino-acid sequence. The GA comprises three steps: selection, crossover and mutation. Through the operation of sequence crossover, it is possible to avoid falling into the local minimum. GAs are used in several fields, especially in informational science, and the results demonstrate the efficiency of GA-based screening. In this study, we evaluated 150 peptides in silico. This represents 1.5% of the total library, and we succeeded in obtaining the peptide that affects α-synuclein aggregation and fibrillation. Therefore, our approach was sufficiently effective for peptide ligand screening. At present, there are many docking simulation softwares, and some of them can perform the calculations more rapidly. Thus, in silico panning has the potential to be applied it to a larger library.Peptide docking simulation takes more time than the docking simulation of small molecules because of the structural flexibility of peptides. Therefore, it is difficult to evaluate the peptide library comprehensively by means of a docking simulation. Yoshimori peptides . Alternaet al. have reported that peptides partially homologous in the NAC region, especially the α-synuclein 60–80 region, bind to α-synuclein across its full length, and that these peptides, modified to dissolve in water, inhibit α-synuclein aggregation[There are some reports of peptides that affect α-synuclein aggregation ,27,28. Tet al. have reported that a peptide designed to inhibit α-synuclein aggregation actually accelerated the aggregation. This peptide lacks one amino acid from the α-synuclein aggregation inhibitor peptide sequence. The protein fibrillation process is assumed to comprise three steps: conformational change, the formation of an amyloid core through self-aggregation, and the elongation of amyloid fibrils. The amyloid-core-forming region is highly hydrophobic. Thus, this region should be isolated to some extent from the solvent. This region may become exposed to the solvent upon the conformational change resulting from the addition of certain factors, such as ligands, which might induce the aggregation of these regions.While there are many reports of aggregation inhibitors, there are some reports of aggregation accelerator peptides ,27,28. TThe ligand with strong binding activity may inhibit aggregation, which is followed by conformational change. Since amyloid fibrils have a very stable conformation, it is thought that the binding ability of self-aggregation is very strong. Weakly bound ligands would not sufficiently inhibit this aggregation followed by conformational change. Therefore, peptides lacking some of the amino acids of the inhibitor peptide sequence would accelerate α-synuclein aggregation and induce fibrillation. In this study, the screened peptides may have bound to the hydrophobic core region, but the binding ability was not high. These peptides might induce conformational changes by binding to the hydrophobic region, and accelerate the α-synuclein aggregation and subsequent fibrillation.in silico panning can be used to screen α-synuclein-binding peptides. Although α-synuclein does not have a defined structure, we were able to screen the peptides by hypothesizing that the target region forms a β-sheet structure. The screened peptides showed low dissociation constants  and affected α-synuclein aggregation and fibrillation. These peptides accelerated α-synuclein aggregation and fibrillation, and may be able to decrease α-synuclein cytotoxicity by decreasing the protofibril amount.We demonstrated that in silico panning approach has a great potential and is applicable to a larger library, it may be useful for the ligand screening of certain disease-related proteins.Since our -1 Å-1. The target peptide, α-synuclein 68–78, was constructed in the same way.We designed the peptides of the initial generation randomly, without bias. Since the target peptide, α-synuclein 68–78, is very hydrophobic, we expected that the peptide ligands would bind to the target peptide through hydrophobic interaction and hydrogen bonding. To avoid an excessively large sequence space, we selected 10 amino acids: glycine, alanine, valine, serine, threonine, proline, glutamine, asparagine, phenylalanine and tyrosine. The molecular operating environment (MOE) from the Chemical Computing Group, Inc. was used for the model building. After the three-dimensional construction of these peptides, in order to optimize these peptides' local conformations, these structures were subjected to an MMFF94 energy minimization protocol until the root mean square of the conjugate gradient reached <0.05 kcal molThe docking simulation described here was carried out using the MOE-Dock default parameters based on the simulated annealing protocol. We selected the MMFF94s force field. The MOE suite utilized here includes an implemented version of the Generalized Born/Surface Area contact function that models the electrostatic contribution to the free energy of the solvation in a continuous solvent model. A docking box was constructed around α-synuclein 68–78. We carried out the MOE-Dock 30 run in our case.We implemented a simple genetic algorithm to produce the next generation. After the docking simulation, the peptides were ranked by their calculated docking energy. The more or less superior peptide sequences were duplicated, and the sequences were recombined at a 1.0 crossover rate. Finally, single-amino-acid mutations were introduced into the peptides at a mutation rate of 5 to 20%. Population sizes of each generation are selected between 20 and 25 according to number of superior peptide.Escherichia coli BL21(DE3) cells transformed the pET28a/human α-synuclein WT plasmid. Following induction with 1 mM isopropyl-1-thio-β-D-galactopyranoside, the α-synuclein was purified by anion exchange chromatography [g for 10 min and lysed in 500 mM NaCl, 20 mM Tris-HCl, pH 8.0, and 1 mM EDTA. The lysate was then boiled for 20 min, and centrifuged for 10 min at 20,000 g. The supernatant was dialyzed against 20 mM Tris-HCl, pH 8.0, loaded onto a ResourseQ 6-ml column (Amersham Biosciences) equilibrated in the same buffer, and eluted with a 0 ~1 M NaCl gradient. The α-synuclein-containing fraction was dialyzed against 10 mM Tris-HCl, pH 7.4.Human wild-type α-synuclein expressed in tography . The bacAll peptides were synthesized using 9-fluorenylmethyoxycarbonyl (Fmoc) chemistry and a solid phase system using the peptide synthesizer PSSM8 . The synthesis resin, TentaGel S PHB-coupled Fmoc-amino acid, was purchased from HiPep Laboratories. Deprotection/cleavage from the resin was performed through treatment with a 90:5:5 (v/v/v) trifluoroacetic acid (TFA)/water/triisopropylsilane mixture for 2 h. Since the glutamine at the N-terminal could undergo a side-reaction, we conducted the acetylation of the N-terminal amine. The synthesized peptides were purified by reverse-phase liquid chromatography using a C18 ODS-column (TOSOH). The mass weights were confirmed by ESI-MS (JEOL).N-hydroxysuccinimide) and EDC (N-ethyl-N'-(dimethylaminopropyl) carbodiimide), coupled with 100 μg/mL of the synthesized peptide in pH-6.0 sodium acetate buffer, and then blocked with ethanolamine. For reference, only activation and protection was carried out, in the same way as mentioned above. The final coupling level of each peptide was approximately 100 RU. Each of the α-synuclein solutions was injected, and at different concentrations a signal increase was observed. The sensor surface was regenerated using 0.5% SDS. The dissociation constants were determined using a Scatchard plot and an equilibrium point.All peptides were immobilized onto the CM5 sensor chip by amine coupling. The CM5 sensor chip (Biacore) was activated by an equimolar mix of NHS (g for 60 min to remove the insoluble aggregates. The peptides were precipitated in the same buffer and mixed with α-synuclein. The α-synuclein and peptide mixtures were incubated in 0.5 ml of the same buffer with 0.02% NaN3 at 37°C by shaking in 1.5 ml tubes. The protein concentrations were kept at approximately 2.0 mg/ml. Fibril formation was monitored by thioflavin T (TfT) fluorescence: 10 μl aliquots were sampled from the incubated samples and added to 1.0 ml of 25 μM TfT in 10 mM Tris buffer, pH 7.4. Fluorescence emission was measured at 485 nm at an excitation wavelength of 450 nm on a FP6500 spectrofluorometer (JASCO). The Rayleigh scattering at 330 nm was used to monitor the total aggregation.All the protein samples were concentrated to approximately 4.0 mg/ml by means of Amicon Ultra-15 filters (Millipore) in 10 mM Tris-HCl, pH 7.4, and centrifuged at 150,000 KA carried out the docking simulation, the determination of the peptide affinity against α-synuclein and the investigation of the effect of the peptides against α-synuclein aggregation and fibrillation. NK carried out the construction of the α-synuclein vector. KS and KI conceived the study, participated in its design and coordination, and helped to write the manuscript. All authors have read and approved the final manuscript.


To evaluate the efficacy and safety of efalizumab in continuous or interrupted therapy of adults with moderate-to-severe plaque psoriasis who had failed to respond to or were intolerant of other systemic therapies, including methotrexate, ciclosporin and psoralen plus UVA phototherapy, or for whom such therapies were contraindicated.Patients received a conditioning dose of efalizumab 0.7 mg/kg followed by once-weekly open-label efalizumab 1.0 mg/kg for 11 weeks. Responders  could continue efalizumab for a further 8 weeks (continuous-treatment period). Nonresponders transitioned to alternative anti-psoriasis medication or stopped treatment. Responders who discontinued efalizumab could restart treatment if symptoms worsened. PGA response was evaluated at Weeks 12 (primary endpoint) and 20, as were the proportions of patients achieving an improvement from baseline of ≥50%, ≥75% and ≥90% in Psoriasis Area and Severity Index (PASI) .A total of 1,255 patients were included in the intention-to-treat population. At Week 12, 68.0% of patients had a PGA rating of “good” or better. Of 688 patients who entered the continuous-treatment period, 79.5% had a PGA rating of “good” or better at Week 20. At Week 12, median improvement in PASI score was 68.4%. PASI 50/75/90 was achieved by 65.5%/35.9%/13.0% of patients at Week 12, and by 78.2%/52.9%/24.3% of responders at Week 20. Of the 127 responders at Week 12 who discontinued efalizumab, 11% experienced rebound and 56.7% relapsed within 8 weeks after stopping therapy. Efalizumab was well tolerated during the study.Efalizumab provided effective control of psoriasis in the majority of patients during the initial treatment period. The high response rates were maintained in initial responders when treatment was continued beyond 12 weeks. Psoriasis is a chronic inflammatory systemic disease ,2, affecEfalizumab is a recombinant humanized monoclonal immunoglobulin G1 antibody that binds to the CD11a subunit of leucocyte function-associated antigen type 1 (LFA-1). It targets multiple stages in the immunopathogenesis of psoriasis: initial T-cell activation, migration of T-cells into dermal and epidermal tissues, and T-cell reactivation ,5. NumerThe current study evaluated the efficacy and safety of efalizumab in the restricted, difficult-to-treat, European-label population (adult patients with moderate-to-severe chronic plaque psoriasis who have failed to respond to or are intolerant to other systemic therapies), and the management of psoriasis rebound and exacerbation during or after efalizumab treatment. It was conducted according to the European Summary of Product Characteristics for efalizumab, which was current during the time the trial was carried out.All patients were aged ≥18 years with a diagnosis of moderate-to-severe plaque psoriasis and had failed to respond, had a contraindication to, or were intolerant of other systemic therapies, including ciclosporin, methotrexate and PUVA.9/L and a platelet count of ≥100 × 109/L. Systemic anti-psoriasis treatments were discontinued before starting study treatment with no washout period. Investigational or biological treatments for psoriasis  were also stopped at least 3 months before study treatment. Patients were not to receive any primary vaccinations within the 14 days before trial entry. Women of childbearing potential were required to use adequate contraception both during the study and for 3 months afterwards. Patients were excluded if they met any of the following criteria: the sole or predominant form of their psoriasis was guttate, erythrodermic or pustular; they had a history of severe allergic reactions to humanized monoclonal antibodies; they had withdrawn from previous efalizumab treatment as a result of lack of efficacy or an adverse event; they were pregnant or breastfeeding; they had a history of opportunistic infections or ongoing uncontrolled infections; they were seropositive for HIV, hepatitis B or hepatitis C; they had been hospitalized for cardiac disease, stroke or pulmonary disease within the last year; they had a malignancy within the past 5 years . Patients with active tuberculosis (TB), a positive chest X-ray or those who had received treatment for TB within 1 year before entry were also excluded; a chest X-ray within 3 months of study treatment was required for patients considered to be at high risk for TB. Written informed consent was obtained from all patients enrolled in the trial.Patients were required to have a white blood cell count of 4–14 × 10This was a Phase IIIb/IV, multicentre, open-label trial . The trial was performed in accordance with the Declaration of Helsinki Guidelines for Good Clinical Practice, with approval by the independent ethics committee/institutional review board for each centre.The trial design is summarized in Responders could continue to receive weekly open-label s.c. efalizumab at 1.0 mg/kg for up to 8 weeks (continuous-treatment period). Nonresponders switched to treatment with an alternative approved anti-psoriasis medications, chosen by their investigator, for up to 12 weeks (transition-treatment period). Patients could choose to discontinue anti-psoriasis medication on completing the first-treatment period regardless of their response at Week 12. These patients entered the observation period and were monitored without treatment for up to 8 weeks or until signs of worsening psoriasis were observed indicating a possible relapse or rebound (in responders) or disease exacerbation (in nonresponders).Patients who responded during the first-treatment period but experienced worsening psoriasis during the observation period could then receive further treatment with weekly open-label s.c. efalizumab at 1.0 mg/kg for 12 weeks (re-treatment period).PGA ,13 and PDisease exacerbation was defined in nonresponders  as worsening of disease to a more inflammatory state than at baseline, and that occurred within pre-existing plaques, at previously uninvolved sites, or as new morphologies of the disease.Adverse events were assessed at every study visit throughout the trial . Adverse events were coded using MedDRA (version 8.1). Reported adverse events were classified as pre-first-treatment period, first-treatment-emergent or post-first-treatment period events based on their onset dates and the dates of first and last “first-treatment period” treatment administration. The severity of adverse events was assessed as mild (the patient was aware of the event or symptom but it was easily tolerated), moderate  or severe . A serious adverse event was defined as an event resulting in death or that was life-threatening, required hospitalization or prolongation of existing hospitalization, resulted in persistent or significant disability or incapacity, was a congenital anomaly or birth defect, or was a medically important condition .The primary efficacy endpoint was PGA response at Week 12, with outcomes also assessed at Week 20. Patients with missing PGA assessments were classified as nonresponders (worst-case imputation). The proportions of patients with a decrease of ≥50%, ≥75% and ≥90% in PASI score from baseline  were calculated at Weeks 12 and 20.Analyses were conducted according to treatment period for the intention-to-treat (ITT) population, which included all patients who received at least one dose of trial treatment and who underwent at least one post-treatment efficacy assessment. Analyses for the safety population, which included all patients who received at least one dose of trial treatment, included analyses of adverse events, safety laboratory outcomes and vital signs. Patients on continuous treatment who received efalizumab in accordance with the protocol  were evaluated as a subset of the whole continuous-treatment population. Analyses for this trial were primarily descriptive in nature and no formal hypothesis testing was performed. Exact 95% confidence intervals (CIs) for proportions were obtained using the PROC FREQ procedure from SAS. The PROC LIFETEST procedure from SAS was used to generate Kaplan–Meier estimates of time to rebound and disease relapse.The trial was conducted at 170 centres across 18 European countries. Between 13 December 2004 and 12 April 2006, 1,266 patients were enrolled in the trial.Patient disposition is shown in The proportion of patients in the ITT population  with a PGA rating of “good”, “excellent” or “cleared” increased throughout the first-treatment period, reaching 68.0% by Week 12 , 79.5%  had a PGA rating of “good”, “excellent” or “cleared” at Week 20 .The ITT population for the continuous-treatment period included the 58 nonresponder patients mentioned previously who, in a deviation from the trial protocol, continued to receive efalizumab up to Week 20. Of these, 29.3% (N = 17) achieved a PGA rating of “good”, “excellent” or “cleared” by Week 20.By Week 12, the median improvement from baseline in PASI score in the ITT population was 68.4% (median PASI score at baseline of 19.55 and 6.00 at Week 12). The proportion of patients in the ITT population with a PASI 50/PASI 75/PASI 90 response increased throughout the first-treatment period to 65.5%/35.9%/13.0% at Week 12 .At Week 20, the median PASI score was 3.90 for patients in the continuous-treatment period and the proportion of patients in the ITT population with a PASI 50/PASI 75/PASI 90 response increased further through the continuous-treatment period to 78.2%/52.9%/24.3% of responders at Week 20 .Of the 135 responders who entered the observation period and therefore had their efalizumab therapy suspended for 8 weeks, 102 subsequently entered the re-treatment period due to worsening signs of psoriasis during the observation period. The proportion with a PGA rating of “good”, “excellent” or “cleared” increased from 20.4% at Week 1 of the re-treatment period to 55.8% at Week 12 .The proportion of patients who transitioned to other anti-psoriasis treatment at Week 12 (N = 68) and who were identified as having a PGA rating of “good”, “excellent” or “cleared” also increased across the transition-treatment period, from 5.9% at Week 1 to 52.9% by Week 12 . ResponsIn total, 135 of 853 first-treatment responders entered the observation period. Out of the 127 evaluable patients, 56.7% (N = 72) relapsed, with a median time to relapse of 56 days, and 11% (N = 14) of the evaluable patients experienced rebound during the observation period.Of the 402 first-treatment nonresponders, 37 (9.2%) experienced disease exacerbation during the first-treatment period. A total of 21 first-treatment nonresponders entered the observation period and 64 entered the transition-treatment period—treatment with an alternative approved anti-psoriasis medication. Of these, 4.8% (N = 1) and 3.1% (N = 2) experienced new or additional exacerbation of their psoriasis during the observation and transition-treatment periods, respectively. Overall, 12.9% (N = 52) of nonresponders experienced disease exacerbation at some point during the trial.The median duration of exposure to efalizumab in the first-treatment period was 78 days (range: 1–134 days); two patients received efalizumab for >100 days. The median cumulative dose was 11.7 mg/kg (range: 1–18 mg/kg). The median duration of exposure to efalizumab during the continuous-treatment and re-treatment periods was 57 days (range 1–266 days) and 78 days (range 1–373 days), respectively.Treatment-emergent adverse events (TEAEs) are summarized in Twenty-two of the 68 patients (32.4%) who received other approved psoriasis therapies  during the transition-treatment period reported TEAEs. Among those events reported by ≥5% of patients, musculoskeletal disorders (11.8%) and skin disorders (8.8%) were the most common, followed by fatigue, feeling cold, influenza-like illness and pyrexia (each 5.9%).During the re-treatment period, a total of 77 TEAEs were reported in 40.7% of patients. The most common TEAEs reported during the re-treatment period were nasopharyngitis (8.8%), arthralgia (3.5%) and back pain (3.5%).Fewer than 5% of patients experienced serious TEAEs during the first-treatment period, with lower incidences reported during the continuous-treatment and re-treatment periods . The mosOne patient, a 71-year-old man, died during the trial. This patient had received a single dose of efalizumab before being withdrawn because of an upper respiratory infection and fever. A month later he experienced fatal cardiopulmonary failure. This event was considered by the investigator as unlikely to be related to trial medication.During the first-treatment period 89 subjects (7.0%) had 134 adverse events that led to treatment discontinuation. Of these events, the most frequent were due to skin and s.c. tissue disorders, general disorders and administration-site conditions, and musculoskeletal and connective tissue disorders .Four malignancies were reported during the first-treatment period: two men (aged 57 and 44 years) with basal cell carcinoma, a 49-year-old woman and a 43-year-old man with squamous cell carcinoma. Only the last case was considered to possibly be related to trial treatment. The patient in question had a history of squamous and basal cell carcinoma. An additional cutaneous squamous cell carcinoma was reported during follow-up. This occurred 6 weeks after the last efalizumab dose given during the re-treatment period and was considered to possibly be related to study treatment.The most frequently reported infection-related TEAEs during the first-treatment period were nasopharyngitis (3.2%), influenza (2.2%) and herpes simplex (1.9%). Infections and infestations were the most common TEAEs in the continuous-treatment and re-treatment periods  and accoWith the exception of two cases of severe influenza in two patients (0.1%) and two cases of severe bronchitis in two patients (0.1%), no other severe infections occurred in more than one patient. There were no reports of TB, opportunistic infection, demyelinating disease or serious thrombocytopenia in any patients during this trial.This prospective, post-approval clinical trial assessed the efficacy and safety of efalizumab therapy in patients across Europe with moderate-to-severe chronic plaque psoriasis who had failed to respond to, had contraindications for, or were intolerant of other systemic therapies. The main strengths of this study were the large patient cohort and the unique study design, which enabled a more accurate reflection of routine clinical practice by allowing patients receiving other anti-psoriasis drugs, those not receiving any treatment, and those restarting treatment to continue follow-up.Results obtained from the initial continuous-treatment section of the trial show that over two-thirds of patients had responded by Week 12 of efalizumab treatment and this high response rate was maintained in responders who continued treatment for up to 20 weeks. These findings are consistent with data obtained from previous pivotal clinical trials, the open-label LATAM trial and longer-term studies ,8–11,15.Following this initial 12-week treatment period, the findings from this study indicate that efalizumab treatment can be discontinued, if alternative therapy is initiated in its place. Of the responders who stopped efalizumab treatment at the end of the initial 12-week period and did not transition to an alterative therapy, 11% experienced rebound. Responding patients who had discontinued and then restarted efalizumab treatment were found to achieve a similar (but slightly reduced) efficacy and tolerability as attained during the initial treatment period.This trial also explored treatment options for patients who do not initially respond to efalizumab therapy. More than half of the nonresponders who entered the transition-treatment period subsequently achieved a response after changing to another anti-psoriatic therapy.The major limitation of this study was the open-label, noncomparative trial design. Indeed, the range of anti-psoriatic therapies permitted across the transition-treatment period, while an accurate reflection of real-life clinical experience, complicates the analysis of treatment effectiveness during this time. It is also difficult to interpret the data obtained during the observation period, since few nonresponders entered this section of the study.The incidence of psoriasis exacerbation in nonresponders was carefully monitored, but found to be lower after the initial 12-week treatment period, indicating that the discontinuation of efalizumab in these patients was not associated with psoriasis-related adverse events. Due to the immunomodulatory mechanism of action of efalizumab, the incidences of infections and malignancies in treated patients were also followed closely. During this trial the safety of efalizumab was found to be acceptable and consistent with previous results. No new safety issues were identified. The incidence of TEAEs was lower in the continuous-treatment period than in the initial-treatment period, with influenza-like symptoms being the most frequent TEAEs in the first-treatment period, as observed in previous studies –10. UnliNo TEAEs related to opportunistic infections were observed during this trial. However, opportunistic infections have been reported in the post-marketing surveillance in patients with psoriasis receiving efalizumab. In particular, cases of JC virus infection resulting in progressive multifocal leucoencephalopathy have been reported in patients receiving efalizumab continuously for more than 3 years. After evaluating all available safety data, the European Medicines Agency concluded that the benefits of efalizumab treatment no longer outweighed the risks associated with the drug and recommended suspension of marketing authorization on 19 February 2009.This study addressed important questions relating to the appropriate management of both responding and nonresponding psoriasis patients in real-life clinical situations, beyond the initial 12-week treatment period. Efalizumab was found to provide effective control of psoriasis in two-thirds of patients within 3 months of treatment. Of 127 evaluable patients who entered the observation period, more than half relapsed and 11% (N = 14) experienced rebound during the observation period. However, the greatest therapeutic benefit was obtained by initially responding patients who were given continuous efalizumab-treatment for the full 5-month trial period.


After more than 200 genome-wide association studies, there have been some successful identifications of a single novel locus. Thus, the identification of single-nucleotide polymorphisms (SNP) with interaction effects is of interest. Using the Genetic Analysis Workshop 16 data from the North American Rheumatoid Arthritis Consortium, we propose an approach to screen for SNP-SNP interaction using a two-stage method and an approach for detecting gene-gene interactions using principal components. We selected a set of 17 rheumatoid arthritis candidate genes to assess both approaches. Our approach using principal components holds promise in detecting gene-gene interactions. However, further study is needed to evaluate the power and the feasibility for a whole genome-wide association analysis using the principal components approach. It is common in candidate-gene or genome-wide association studies to perform single-gene association analysis. However, after more than 200 genome-wide association studies (GWAS), there have been fewer novel loci identified than expected , possiblp < 0.001) were excluded from all analyses, leading to a final set of 135 SNPs. Before analysis, the computer program MACH [All of our analyses utilized genotype data of the 868 cases and 1194 controls in the NARAC data set. Analyses were carried out on a set of 17 candidate genes, selected on the basis of a literature search. The candidate genes used in these analyses are listed in Table glk be the number of minor alleles at SNP k for lth subject, l = 1, ..., N, k = 1, ..., K. 2) Calculate the correlation matrix R, where Rij = cor and gi and gj represent the genotypes of all subjects for SNP i and SNP j, respectively. 3) Decompose R by singular value decomposition: R = AΛAT 4) Determine the factor loading by GA, where G is the standardized N × K matrix of genotypes. The standardized genotypes are calculated as: This approach was proposed by Gauderman et al.  to test R and the computer program Haploview [Then, we use PCs that explain at least 80% of the variation as the gene representation to perform a gene-gene interaction analysis, by applying logistic regression to test for interaction between every combination of two PCs. Once significant PC interactions are identified, PC loadings may be used to determine the influence of a specific SNP on the PCs because the loading represents the correlation of a SNP with a component. For better visualization of the gene-PCs and their SNPs position with the LD block plots, we created a graphical display using our own function in the statistical package Murcray et al.  proposedg1, g2) based on the approximate method to screen for epistasis implemented in PLINK [g1 and g2 as 0, 1, or 2, representing the number of minor alleles. A χ2 with 1 degree of freedom is used to test the association between each pair of SNPs. Pairs of SNPs are selected for analysis in Step 2 if they exceed a given significance threshold, p <α*. In our case, we selected α* = 0.05.For each pair of SNPs, we perform a test of association between the two SNPs  and controls (D = 0). An interaction is considered significant when the p-value of interaction  is less than or equal to α/M, where α = 0.05.where Q values [p-value < 5.5 × 10-6 /2 with K = 135 SNPs) compared with the two-stage approach with p-value in the first stage < 0.05 and a p-value in the second stage < 3.2 × 10-5 .Figure Q values . Figure Q-value only. The strongest interactions were observed within the HLA region with TNF-PC3 and VEGFA-PC1 and HLA-C-PC1 and TNF-PC2 . Outside the HLA region we observed two moderate interaction effects involving STAT4-PC5 and C5-PC4, and TNF-PC2 and STAT4-PC5 . Figure STAT4 and C5, and the linkage disequilibrium (LD) blocks within these genes. The STAT4-PC5 interaction contains four SNPs with absolute value of loadings ≥ 0.5 and they represent their own block. The C5-PC4 interaction contains three SNPs with loadings ≥ 0.5, where the two SNPs (rs10760131 and rs10985112) with the two highest loadings, 0.9 and 0.8 respectively, belong to the same block. On the other hand, the two-step approach detected only interactions within the HLA region. The strongest interactions were between SNPs of VEGFA with HLA-C, LTA with HLA-B, and with HLA-C, and HLA-B, with HLA-C. Code to perform these analyses is available from the authors by request.The PC approach detected several PC interaction effects when using We extended two approaches previously used for gene-level tests or gene-environment interaction analysis to screen for gene-gene interactions in 17 candidate genes for RA using the GAW16 NARAC data. In the PC approach we calculated the SNP loadings for each PC and viewed them in the context of the gene LD structure generated using Haploview Figure . This coα* in Stage 1. Recently, a similar two-step method was proposed and shown to be more powerful than a one-step approach [We could not identify any significant interactions using the two-step approach. There are several possibilities, including the elimination of SNPs with low allele frequency, and the choice of approach . FurtherUsing PCs is a promising approach to screen for potential interactions. As shown in our results, it can detect interactions not observed based on SNP-SNP interactions assessed using either a single-step or a two-step approach. Furthermore, the method used to correct for multiple comparison also plays an important role.GAW16: Genetic Analysis Workshop 16; GWAS: Genome-wide association studies; LD: Linkage disequilibrium; NARAC: North American Rheumatoid Arthritis Consortium; PC: Principal components; SNP: Single-nucleotide polymorphismThe authors declare that they have no competing interests.JL carried out the candidate genes selection, programming, and performed the statistical analysis. RT participated in the candidate genes selection and programming. JMB participated in the design of the study and helped to draft the manuscript. MdA conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum, is currently evaluated as a fixed dose combination with artesunate for the treatment of uncomplicated malaria. In this study, the in vitro activity of pyronaridine against clinical isolates of P. falciparum from Lambaréné, Gabon, was assessed in order to obtain baseline data on its activity prior to its future use in routine therapy. Moreover, follow-up assessment on the in vitro activity of chloroquine, artesunate and quinine was performed.Pyronaridine, a Mannich base anti-malarial with high efficacy against drug resistant In vitro response of field isolates of P. falciparum to pyronaridine, chloroquine, artesunate and quinine was assessed using the traditional WHO microtest. In addition, the histidine-rich protein 2 (HRP-2) assay was performed and evaluated for its future implementation for follow-up of drug susceptibility testing.in vitro activity against P. falciparum, with a geometric mean cut-off concentration of 9.3 nmol/l. Fifty percent effective concentrations were 1.9 nmol/l and 2.0 nmol/l in the WHO microtest and HRP-2 assay, respectively. Results matched closely in vivo findings from a recent clinical trial on pyronaridine-artesunate treatment. One isolate showed diminished sensitivity to artesunate. For chloroquine and quinine resistance levels were comparable to prior studies from Lambaréné. Results from the novel HRP-2 assay corresponded well to those obtained by the WHO microtest.Pyronaridine exhibited a high Pyronaridine is highly active in chloroquine-resistant parasites and seems a promising partner drug for artemisinin-based combination therapy in Africa. Plasmodium falciparum in routine surveys, the evaluation of new compounds against field isolates is of major importance for drug development.Malaria continues to be a major cause of morbidity and mortality in sub-Saharan Africa, particularly in young children. Early detection and effective chemotherapy remain the cornerstones in its control . The rapin vitro drug sensitivity under field conditions – has been used for this purpose [In Lambaréné, Gabon, routine anti-malarial drug susceptibility monitoring has been performed since 1992 . So far  purpose . Meanwhi purpose . This me purpose .P. falciparum in vivo [The anti-malarial agent pyronaridine is a Mannich base derivative of mepacrine, one of the earliest synthetic anti-malarials . It is c in vivo ,11, yet  in vivo .P. falciparum field isolates from Lambaréné to pyronaridine in order to obtain baseline data on the activity of this for Africa yet novel anti-malarial drug prior to its widespread use in routine therapy. In addition the study was designed to assess the potential for the novel HRP-2 assay to replace the standard WHO microtest.The present study aimed to assess the susceptibility of clinical P. falciparum malaria transmission [P. falciparum monoinfection with 1,000–100,000 asexual parasites per μl blood, no schizontaemia, no signs or symptoms of severe malaria, and no history of intake of anti-malarial drugs in the preceding month. Information about age, sex, and duration of fever was collected on a questionnaire. Informed consent was obtained from participants or their legal representatives. The study was approved by the Ethics Committee of the International Foundation for the Albert Schweitzer Hospital in Lambaréné.The study was carried out at the Medical Research Unit of the Albert Schweitzer Hospital in Lambaréné, Gabon, in a region of stable, hyperendemic smission ,14. PatiP. falciparum to pyronaridine, artesunate, chloroquine and quinine. First, the standard World Health Organization in vitro microtest was used similar to previous reports, measuring drug-dependent inhibition of schizont maturation (SMI) within 24 hours by microscopic assessment [Two different methods of drug sensitivity testing were deployed in order to assess the susceptibility of fresh field isolates of sessment . Brieflysessment .Fifty μl BMM were transferred into scheduled wells and incubated at 37.5°C in candle jars. After 24 hours parasites were harvested and Giemsa-stained thick blood films were prepared. The number of mature schizonts was microscopically counted against 200 asexual parasites in each well. Tests were considered successful if at least 10% schizont maturation was observed in the drug-free control-well.in vitro parasite-growth, a thick blood smear of one control-well was performed after 26 h. One non-treated 26 h sample was frozen to calculate background HRP-2 production. Parasite culture was judged successful if at least 10% parasites matured to schizonts at the 26-hour time point. After 72 h plates were freeze-thawed twice. Parasite growth, calculated from HRP-2 levels, was measured with an enzyme linked immunosorbent assay at an absorbent maximum of 450 nm.In addition, the HRP-2 assay was performed according to the published standard operational procedures . Two ml Non-linear regression analysis with 4-parameter fits of log-concentration/response curves was used to determine individual inhibitory concentrations of the respective isolates. All regressions were checked manually. Cut off concentrations were calculated as geometric mean of the lowest individual concentrations with no mature schizont among 200 parasites in the WHO microtest. Nonparametric analysis was used for concentration data that was not normally distributed. A two-tailed Mann-Whitney-U-Test was performed in order to test for difference between the two drug sensitivity assays. All tests were performed at a two sided significance level of α = 0.05.Ninety-five outpatients attending the Albert Schweitzer Hospital were included in this study. The patients' median age was three years, ranging from three month to 18 years, and 55% were female. Median asexual parasitaemia at presentation was 36,500 per μl blood. In the schizont maturation microtest 32, 36, 34, and 32 out of 95 isolates yielded valid results for pyronaridine, chloroquine, artesunate and quinine, respectively. Twenty-five isolates fulfilled the criteria for successful parasite-culture in the HRP-2 assay and 15, 16, 12 and 14 were successfully employed for drug sensitivity testing, respectively. Post-hoc analysis of the unexpected low success rate of cultivation, especially in the HRP-2 assay, revealed that poor growth was associated with one lot of the commercially acquired medium. Those isolates were identified and excluded from the analysis. Table in vitro growth and schizont maturation in the WHO assay at a geometric mean cut-off concentration of 9.3 nmol/l BMM. The respective geometric mean EC99 concentration in the HRP-2 assay was 9.0 nmol/l. In one isolate schizont maturation was observed at a pyronaridine concentration of 122 nmol/l BMM, resulting in a cut-off concentration of 366 nmol/l BMM. Further two isolates showed cut-off concentrations of 122 nmol/l. Geometric mean EC50 values were 1.9 and 2.0 nmol/l BMM for the WHO microtest and the HRP-2 assay, respectively.Pyronaridine inhibited 99 value in the HRP-2 assay. The most sensitive isolate showed a cut-off concentration of 3.2 μmol/l blood, all other isolated exhibited cut-off concentrations of 6.4 μmol/l blood or higher. In four parasite cultures, microscopic assessment revealed schizont maturation up to the highest concentration of 51.3 μmol/l blood. The 50 percent effective concentrations were 7.1 μmol/l blood in the WHO microtest and 5.5 μmol/l in the HRP-2 assay, respectively.The geometric mean cut-off concentration for chloroquine was 21.7 μmol/l blood in the WHO assay, 19.2 μmol/l blood was the respective ECin vitro schizont maturation was not inhibited by the highest artesunate concentration of 85 nmol/l BMM. The same isolate exhibited good sensitivity to pyronaridine and to quinine. The geometric mean cut-off concentration was 9.2 nmol/l BMM. The HRP-2 assay showed a mean EC99 level of 15.2 nmol/l BMM.Fifty percent effective concentrations were 2.1 nmol/l BMM in the microtest and 2.3 nmol/l BMM in the HRP-2 assay for artesunate. Two isolates exhibited schizont maturation at 28.6 nmol/l BMM, yielding cut-off concentrations of 85 nmol/l BMM. In one isolate 99 in the HRP-2 assay was 662 nmol/l BMM. The EC50 values in the WHO microtest and in the HRP-2 assay were 272 and 204 nmol/l BMM, respectively.All isolates were susceptible to quinine. Highest cut-off concentrations were at 1783 nmol/l BMM, thus well below the threshold of resistance (5120 nmol/l BMM). The geometric mean cut-off concentration was 873 nmol/l, the EC50 values of the individual isolates was performed in order to assess the relationship between sensitivity of parasites to pyronaridine, chloroquine, artesunate and quinine . Interestingly, the activity of pyronaridine was correlated with artesunate (r = 0.84 p < 0.0001), but not with chloroquine or quinine.Correlation analysis of ECne Table . Signifi50 values from the two drug sensitivity assays were found in U-test of pooled data of the individual drugs . Differences in geometric means of EC50 values were largest for chloroquine  and quinine . Figure 50 values for the respective drugs.No statistically significant differences between the EC99 values of the HRP-2 assay  [Pyronaridine exhibited a high level of e assay) ,16.99 in HRP-2 assay) and 9.0 nmol/l (cut-off concentration in WHO microtest) inhibited growth of P. falciparum in vitro. In a recent pharmacokinetic assessment of a fixed-dose pyronaridine artesunate combination in African children, the mean maximal plasma concentrations of pyronaridine were 115 nmol/l in patients [in vivo therefore occurred only after plasma levels fell below concentrations similar to cut-off concentrations obtained in this in vitro study.Median pyronaridine concentrations of 9.3 nmol/l  were comparable to results obtained in 2002 (286 nmol/l) [As in previous reports from Lambaréné, susceptibility to quinine was high in this study. Mean ECComparative assessment of the HRP-2 assay and WHO microtest was performed in order to establish baseline data for the use of the ELISA based assay in Lambaréné in the future. Results obtained by the HRP-2 assay closely paralleled those obtained by the WHO microtest despite methodological problems in this study due to a deficient batch of culture medium. Considering its reduced labour-intensity and good reproducibility the HRP-2 assay can, therefore, be recommended for follow up of anti-malarial drug susceptibility testing and will replace the WHO microtest in future assessments at our study site.in vitro drug activity was similar, compared to data from earlier in vitro assessments, and results of the HRP-2 assay were comparable to those obtained by the traditional WHO microtest.Artesunate, chloroquine and quinine P. falciparum and corresponded well to recent findings of pyronaridine anti-malarial activity in vivo. Pyronaridine is recommended for further clinical development in combination therapy and continued in vitro drug activity monitoring.The study demonstrated high anti-malarial activity of pyronaridine against fresh field isolates of The authors declare that they have no competing interests.FK contributed to the conception and design of the study, performed parasite cultivation and microscopic assessment, analysed the data and wrote the manuscript. PP performed parasite cultivation and HRP-2 immunoassay. SB gathered field isolates from patients and performed parasite cultivation. BM contributed to performance of immunoassay and analysis of data, PGK revised the manuscript and supervised the research group, MR conceived and designed the study, contributed to analysis of data, performed second reading for microscopic assessments in WHO microtest and drafted and revised the manuscript. All authors read and approved the final version of the manuscript.